Journal of Infection 82 (2021) e35–e37
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Journal of Infection
journal homepage: www.elsevier.com/locate/jinf
Letter to the Editor
Ct value is not enough to discriminate patients
harbouring infective virus ✩
Dear Editor,
In reference to the letter to the editor by Krupp et al.1 we have
some data of interest obtained from our SARS-CoV2 culturing ex-
perience.
RT-PCR has become in the more sensitive method to detect
SARS-CoV-2 infection. Amplification of genomic sequence is mea-
sured in cycle thresholds (Ct). Despite its high sensitivity and
wider application, RT-PCR has an important limitation. It does not
distinguish between infectious and non-infectious virus. Different
studies regarding virus shedding in mild and severe COVID-19 pa-
tients during hospitalization showed that SARS-CoV-2 RNA may
well be detected in the respiratory tract for up to 21, 32 and 34
days, respectively2-4 .
Also, viral loads on different time courses of SARS-CoV-2 infec-
tion could show changes in disease stages. It was reported that vi-
ral load was high in the early and progressive phase of the dis-
ease and decrease gradually in the recovery phase, peaking at 4
to 6 days after onset of symptoms and gradually decline after-
wards. However, initial viral load was not correlated with days af-
ter symptom onset. Nowadays the use of Ct values as direct mea-
sure of SARS-CoV-2 viral load should be taken with care because
is not a standard measure of viral load in clinical samples, as it
was demonstrated by Dahdouh et al.5 .. Finally, the prolonged vi-
ral shedding is relevant for the control of infection in hospitals
and discharge management, the correlation between detectable vi-
ral RNA and virus isolation in clinical samples remains unclear
though4 .
Viral transmission and infectivity are one of the most important
determinants for prevention strategies in respiratory viral infec-
tions and can be substantially reduced by containment measures
such as isolation and quarantine. Knowledge of SARS-CoV-2 viral
shedding is of primary importance for the development of effective
prevention and control measures. We investigated the infectivity of
clinical samples obtained from patients with SARS-CoV-2, compar-
ing the results obtained by RT-PCR with the growth capacity of the
virus in vitro.
For all patients, 400 μL of nasopharyngeal swab fluid (NP)
(UTM R
: Viral Transport, Copan Diagnostics, Inc., Murrieta, CA)
were obtained. Samples were inactivated with 400 μL of AVL
buffer (Qiagen) at a 1:1 ratio (400 μl AVL: 400 μl nasopharyngeal
swab fluid) and incubated for 10 min at room temperature before
✩
SARS-CoV-2 Working Group: A. Gutiérrez-Arroyo, I. Bloise, F. Lázaro-Perona, M.I
Quiles-Melero, G. Ruiz-Carrascoso, I. Falces-Romero, M. Ruiz-Bastián, C. Toro-Rueda,
S. García-Bujalance, B. Gómez-Arroyo, C. García-Sánchez, V. Guedez-López, M. Gracia
Liras-Hernández, M. Sánchez-Castellano, P. García-Clemente, P. González- Donapetry.
https://doi.org/10.1016/j.jinf.2020.11.025
0163-4453/© 2020 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
nucleic acid extraction. Non-inactivated fractions were conserved
at −80 °C for viral growth assays.
MagMax Express 96 (Applied BiosystemsTM , MA, USA) was used
for nucleic acid extraction according to manufacturer’s instructions.
TaqMan2019-nCoV Assay Kit v1 (ThermoFisher, MA, USA) was used
for detection of viral RNA.
Those samples conserved at −80 °C were re-evaluated using the
same methods extraction and RT-PCR kit after a freeze-thaw cycle
before viral growth assays in cell culture.
All procedures for viral culture followed the laboratory
biosafety guidelines and were conducted in a biosafety level 3 fa-
cility. 250 μl of nasopharyngeal swab fluid (NP), freshly obtained
or conserved at −80 °C, were inoculated in confluent Vero E6
cells monolayers (ATCC CRL-1586) in 250 μl of Minimum Essen-
tial Medium culture medium with 4% fetal calf serum and 1% glu-
tamine. Vero E6 cells were growth in Dulbecco’s Minimal Essential
Medium (DMEM) supplemented with 10% fetal bovine serum and
Antibiotic-Antimycotic GibcoTM solution and in a humidified 37 °C
incubator with 5% CO2 .
After 4 days infected cells were fixed with 10% formaldehyde
in phosphate-buffered saline (PBS) for 30 min at room temperature
and then stained with crystal violet and observed to confirm cyto-
pathic effect (CPE). Culture supernatants were collected from each
well, for RNA extraction and SARS-CoV-2 RT-PCR.
A cell culture is suspected of hosting virus replication based on
the presence of CPE including damage to the monolayer, cell clear-
ing and morphological changes, and if the RT-PCR was positive and
the Ct was at least 2 cycles lower than that of the original sample.
In all the studied samples, the presence of other microorganism
that could produce a cytopathic effect was ruled out after perform-
ing the multiplex PCR FTDTM Respiratory pathogens 21 (Fast Track
Diagnostic. Siemens, Madrid, Spain).
A total of 72 specimens of NP from 66 patients were analysed in
this study. Medical records for these patients regarding epidemio-
logical and demographic characteristics, symptom history and rel-
evant exposure data on admission were retrospectively reviewed.
Sixty-six samples were positive and six were negative for SARS-
CoV-2 genome by PCR. A total of five (7.57%) asymptomatic, forty-
six (69.69%) mildly symptomatic and fifteen patients (22.72%) with
severe pneumonia due to SARS-CoV-2 were identified. Seven pa-
tients died due to severe pneumonia by SARS-CoV-2. The average
age of the patients was 52.64 years (range, 25 to 89), and 42 pa-
tients were male (63.63%).
Fever was present in 31 out of the forty-six mildly symptomatic
patients (31/46, 67.39%), and cough was present in 29 of them
(29/46, 63.04%).
Five patients had neither clinical symptom (5/66, 7.57%), two of
them were close contacts of positive confirmed cases, two were
previous positive cases of SARS-CoV-2 PCR and the fifth case was
M.P. Romero-Gómez, S. Gómez-Sebastian, E. Cendejas-Bueno et al.
Fig. 1. Samples without freeze-thaw.
detected in a routine screening prior to hospital admission due to
digestive bleeding.
Specimens collected between February and April (44 out of 72)
were stored at −80 °C until being analysed for SARS-CoV-2 vi-
ral growth. Samples collected between May and June (28 out of
72) were conserved for no more of 48 h at 6 °C until virus cul-
turing. Samples collected during the first period had lower SARS-
CoV-2 targets Ct than those obtained during the second period
(p = 0.0 0 02). We obtained 17 viral isolates of SARS-CoV-2 (25.75%)
from inoculated Vero E6 cells showing CPE, 14/44 (31.81%) from NP
that underwent a single freeze-thaw cycle (first period) and 3/28
(10.71%) of samples without a freeze-thaw cycle (second period).
The positive cultureś rate was higher in samples with a freezing-
thawing cycle (P value= 0.0225). Infection by other respiratory mi-
croorganism was not detected.
We also compared the RT-PCR Ct of the culturable and noncul-
turable samples. We found a significative difference between Ct of
culturable and nonculturable samples (p< 0.0 0 01). The average Ct
values in the specimens with culturable virus were 25.97±4.59 for
ORF1ab, 25.43±4.58 for gene N and 25.54±5.1 for gene S in sam-
ples previously freezed and thawed, and 23.69±0.88, 23.27±0.66
and 23.06±1.28, respectively, in fresh samples (Figs. 1 and 2).
The highest Ct value in samples with positive cultures was
found to be 36.08, 37.73 and 37.41 for the ORF1ab, N and S
genes, respectively in a specimen previously freeze-thawed from
a woman with cough and fever. This sample had been taken one
day after symptoms onset.
The average time elapsed since the onset of symptoms to sam-
ple collection was 7.05 days in patients with positive viral growth
(range:0–12 days) and 13.51 days (range: 1–106 days) in patients
without viral growth (p< 0.0 0 01).A positive or negative RT-PCR re-
sult might not be enough for an accurate identification of the in-
fectious potential of the patients. The best indicator of infectious-
ness is the viral culture of samples from patients with SARS-CoV-
26 , 7 . Different studies show that there is an inverse relationship
between Ct, viral culturability and date of symptom onset8 , 9 .
36
Journal of Infection 82 (2021) e35–e37
Fig. 2. Samples freeze-thaw.
The duration of viral shedding in NP evaluated by RT-PCR for
SARS-CoV-2 as a determining factor to recover the virus in culture.
The range reported was between 7 and 35 days after symptom on-
set4 . In our study, the maximum time after the onset of symptoms
in which the virus was isolated was 12 days.
Positive cultures were associated with low Ct values. Kujawski
et al. obtained viral growth from NP samples with PCR Ct values
between 17.0 and 39.0. In the work of Singanayagam et al. the RT-
PCR cycle threshold median values was 31.15, and the estimated
probability of recovery of virus from samples with Ct>35 was 8.3%.
In our series, we obtained viral growth in samples with Ct between
21.54 and 37.73 in gene N, which agrees with previously published
data4 , 10 .
On the other hand, we studied the effect of storing the samples
at −80 °C and the subsequent thawing on the viability of SARS-
CoV-2. The results suggest that a freeze-thaw cycle did not signifi-
cantly affect the positive culture rate as previously described6
In conclusion, Cts values cannot be used as unique tool to iden-
tify those patients who can be infective despite a positive SARS-
CoV-2 PCR. Different studies demonstrated that viral growth could
be obtained with high Cts. Our results also suggest that the mo-
ment of sample collection after onset of symptoms is an important
variable that has to be taken into account.
References
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María Pilar Romero-Gómez∗
Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ,
Paseo de La Castellana 261, Madrid 28046, Spain
37
Journal of Infection 82 (2021) e35–e37
Silvia Gómez-Sebastian
Department of Preventive Medicine and Public Health and
Microbiology, C/ Arzobispo Morcillo, 2, Madrid 28029, Spain
Emilio Cendejas-Bueno, María Dolores Montero-Vega,
Jesús Mingorance, Julio García-Rodríguez
Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ,
Paseo de La Castellana 261, Madrid 28046, Spain
∗ Corresponding author.
E-mail address: mpromero.hulp@salud.madrid.org
(M.P. Romero-Gómez)